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Vol. 6, 1463-1476, November 1995 Cell Growth 1463 & Differentiation Modulation of Retinoblastoma and Retinoblastoma-related Proteins in Regenerating Rat Liver and Primary H epatocytes’ Guangsheng Fan, Ruiling Xu, Martin W. Wessendorf, Xiaoming Ma, Betsy T. Kren, and Clifford J. Steer linked to its simultaneous suppression of cell cycledependent kinase 4 and cyclin E protein levels. Departments of Medicine 1G. F., R. X., X. M., B. T. K., C. J. 5.1 and Cell Biology and Neuroanatomy IM. W. W., C. J. SI, University of Minnesota Medical School, Minneapolis, Minnesota 55455 Introduction Abstract Protein expression of the retinoblastoma (Rb) tumor suppressor gene product was examined by immunoblot analysis of nuclei isolated from regenerating rat liver after 70% partial hepatectomy (PH). Levels were almost undetectable in quiescent 0-h livers but increased 1 5- to 60-fold 3 to 24 h post-PH, 1 05-fold at 30 h, and 20- to 50-fold at 60 to 72 h post-PH. Expression returned to near baseline levels at 1 8, 42, and 48 h post-PH. A similar pattern of Rb protein expression in the regenerating liver was observed by indirect immunofluorescence microscopy, with peak nuclear expression at 30 h post-PH. Rb-related proteins with apparent molecular masses of 300, 1 56, and 74 kDa were detected in regenerating liver using mAbs to the Rb protein. Their expression increased 6- to 8-fold during regeneration, and only p1 56 returned to baseline levels at 60 h post-PH. Rb and its related proteins were detected in cultured primary hepatocytes, and although total protein levels did not change appreciably, there was a dramatic shift from cytosol into nuclei through 96 h. The half-life of the Rb protein was determined to be 1 .9 h in regenerating liver and 2.2 h in cultured primary hepatocytes. Rb protein abundance in synchronized HuH-7 human hepatoma cells was cell cycle dependent and exhibited peak nuclear expression during S phase. Rb protein was detected primarily in its hyperphosphorylated state during liver regeneration and through the cell cycle of the HuH-7 cells. In vivo administration of transforming growth factor 31 an inhibitor of DNA synthesis in regenerating liver, resulted in reduced expression of Rb as well as its protein partners, cell cycle-dependent kinase 4 and cyclin E. The results suggest that in the regenerating rat liver and in synchronized HuH-7 cells, expression of Rb protein is modulated in a cell cycle-dependent fashion, remains primarily in a hyperphosphorylated state, and exhibits a relatively short half-life. The inhibition of Rb protein expression by transforming growth factor f31 may be , Received 5/19/95; 1 This study was Foundation 2 To whom revised supported )to C. J. S.). requests for 7/14/95; in part reprints accepted 9/1/95. by a grant from should Medicine, UMHC Box 36, University Delaware Street SE, Minneapolis, MN (612) 625-5620. be the addressed, of Minnesota 55455. Phone: Minnesota The Rb3 gene is one of the better characterized members of the tumor suppressor gene family. It was originally identified and eventually cloned by virtue of its absence in a number of Rb tumor cell lines (1-3). Subsequent studies revealed that inactivation of the Rb gene was a frequent event in tumomigenesis. Rb is known to play a key mole in the regulation of cell proliferation (4), and it now appears to also be involved in the induction of the fully differentiated state. For example, it has been suggested that Rb protein, in association with myogenic factors such as Myo D, is mequmred to bring about terminal differentiation of muscle cells (5). The Rb gene encodes a nuclear phosphoprotein of 1 10 kDa, which is present in most cell types and which also exists in phosphorylated states ranging in size from 1 10 to 1 1 6 kDa (6). It is now well established that activity ofthe Rb protein during the cell cycle is regulated by its level of phosphomylation. It is underphosphomylated in G ; hyperphosphorylated at the G1-S phase transition; remains phomylated in 5, G2, and most of M; and reverts undemphosphorylated Phosphomylation (7-9). regulated Whereby state at on before of the Rb the M-G0 protein appears phosto an transition to be at the level of both cell growth and differentiation. mitogenic stimulation activates Rb protein phos- phomylation, signals of differentiation are associated with dephosphomylation of the protein (8). In short, unphospho- rylated Rb suppresses cell proliferation and promotes cellulan differentiation in contrast to phosphorylation, which inactivates Rb activity and allows the cell to enter S phase. An important property in defining its binding domains has been the ability of the functional Rb protein to form stable complexes with the E1A protein of adenovirus (10), the large T antigen of polyomavmmus (1 1), and the E7 protein of papillomavinus (1 2). Any mutation that affects the ability of these oncoproteins to bind Rb dramatically reduces the transformation potential of these small DNA tumor viruses (1 1 , 1 3). Each of the oncoproteins contains a short homologous, colineam sequence, which is thought to serve as the Rb-binding motif (14). Interestingly, that binding site maps to the region of the Rb protein that is most frequently mutated in tumors (1 5, 1 6). Taken together, the results suggest that these DNA tumor viruses may stimulate cellular proliferation by binding to and sequestering Rb protein in a manner that mimics its loss in naturally occurring tumors. Medical at Department of Medical School, (612) 625-8999; 516 Fax: 3 The abbreviations used are: Rb, retinoblastoma; BndUrd, 5-bromo-2’-deoxyunidine; CDK, cell cycle-dependent kinase; PH, partial hepatectomy; TGF-l transforming growth factor l ; FBS, fetal bovine serum; kDa, kilodalton(s). , 1464 Rb Protein Expression Binding in Regenerating Rat Liver to Rb as a necessary induced by viral Rb protein-bound proliferation questening proteins step oncoproteins in the suggests displace cellular proteins required for normal cell and/on differentiation. By binding to and seRb in its undemphosphorylated state, the oncoare thought to release the growth posed by Rb and allow for uncontrolled date, a large group of cellular proteins that transformation that they bind Rb, including those restraints im- cell growth (4). To have been identified involved in transcriptional regulation such as E2F (1 7, 1 8), Myo D (5), Pu.1 (1 9), c-Myc (20) and ATF-2 (21), the signal transducing protein p48 (22), the structural protein lamin C (23), and specific kinases cellular proteins The level remains of Rb expression to be fully appears characterized. to be critical in de- tenmining the status of cell growth. Overexpression of Rb by DNA transfection on microinjection of protein results in inhibition of cell growth under conditions in which the wild-type cell proliferates normally (28). The two populations of cells may represent the difference in cell function associated with a critical threshold of Rb protein. In fact, titration of Rb protein results first in a gradual decrease in cell cycle activity and then a sudden absence of activity, as ifa specific level ofpmotein is required to maintain cell cycle arrest (29). Interestingly, the Rb protein down-regulates its own promoter activity (30), suggesting that a threshold effect may exist in the control of cell growth by Rb. In an effort to further characterize its expression during the cell cycle, we have investigated the abundance and state of phosphonylation of Rb protein in regenerating liven and primary hepatocytes. Liven regeneration after 70% PH is a well-characterized in vivo model of cell replication (31). Growth ofthe liver is triggered by the decrease in mass and involves a synchronized and highly regulated prolifemation of cells in the remaining lobes. There is an initial peak of DNA synthesis which is followed 33). A second, by hepatocytes 6 to 8 h later less intense at 20 to 24 h post-PH, by a wave of mitosis (32, peak in DNA approximately 48 h post-PH and reflects manly of nonpanenchymal cells. However, ported recently that neously in cells all the G0-G, of the transition liven and synthesis occurs replication, pniit has been me- occurs that simulta- G, of the nonparenchymal cells is simply prolonged (34). In the young adult mat, the original hepatic mass is usually attained 1 0 to 1 2 days after 70% PH. Once the original mass is restored, the cells of the liver resume a quiescent state. Multiple panacnine and autocnine factors participate in the finely single trolling orchestrated regulation of liver regeneration; and no factor has been identified as the master switch coninitiation and termination of growth. There is a sequential and regulated and delayed-early growth ing liven after PH (34, 35). expression of many immediateresponse genes in the negenematThese factors appear to partici- pate in the transition of cells from quiescence into G. Because of these characteristics, the regenerating liven after PH provides a unique system to study Rb regulation of cell proliferation and differentiation. The present study extends the analysis of Rb transcript modulation In addition, protein in Immunoblot tion were expression of Rb protein is modulated in a cell cycle-dependent fashion and remains primarily in a hypemphosphorylated state. Furthermore, nuclear expression of the Rb protein during liver regeneration is significantly uncoupled from transcript expression through at least 72 h. In addition, the inhibition of DNA synthesis in the regenerating liven by TGF-31 is associated with suppression of Rb abundance as well as CDK4 and cyclin E, two key modulators of Rb phosphonylation. (24), phosphatases (25), and cyclins (26, 27). However, the biological significance of the interaction of Rb protein with these phorylated species. The results suggest that in the negenerating mat liven and in synchronized HuH-7 cells, expression in the regenerating rat liven after 70% PH (36). it investigates the cell cycle expression ofthe Rb synchronized HuH-7 human hepatoma cells. analysis and immunohistochemical localizaused to determine the subcellulan distribution and pattern of the Rb protein and its various phos- Results Retinoblastoma Protein Expression in Regenerating Rat Liver. In this study, expression of the 1 1 0-kDa Rb tumor suppressor gene product was characterized in regenerating mat liven after 70% PH. Nuclei were isolated at varying time points from post-surgery sham and regenerating livers through and analyzed for Rb protein expression 72 h using mAbs to several different epitopes of the protein. XZ1 61 provided the strongest signal and was used primarily throughout the study. Protein levels were almost undetectable in 0-h liver and remained so until 3 h post-PH, when theme was an increase to 50-fold by 1 2 h (Fig. 1 , A and B). An abrupt decrease in expression was observed at 1 8 h post-PH, followed by a 55- and 105-fold increase above control levels at 24 and 30 h post-PH, respectively. Expmession decreased to near baseline levels at 42 and 48 h post-PH and again significantly increased 20- and 50-fold at 60 and 72 h post-PH, respectively. By 96 h post-PH, Rb expression returned to near 0-h values. In addition, protein levels remained almost undetectable in sham-operated liv- ens through 72 h (data not shown). Throughout the period of regeneration, the Rb protein remained primarily in the hyperphosphonylated state (Fig. 1 C, ppRb). However, during G,, theme was a notable increase in the hypophosphorylated Rb species until 12 h post-PH, when the ratio returned to baseline levels and 90% of Rb was detected in a hypemphosphorylated state. A Northern blot is provided for cornpanison (Fig. 1A), based on the previous report that rat liver expresses two Rb transcript species 2.8 and 4.7 kb in length (36). Again, the switch in transcript expression occurred at 3 to 6 h in which the 2.8-kb species was reduced approximately 95% and that of the 4.7-kb species increased ap- proximately protein 15-fold oven baseline expression occurred by immunoblot, in transcript expression levels. In contrast no significant to Rb changes at 24 to 30 h post-PH on 60 to 72 h. To confirm the analyses in tissue, staining provided The results obtained from the immunoblot we used indirect immunofluomescence with the same mAbs to Rb (Fig. 2). Again, XZ161 the strongest signal and least nonspecific staining. results analysis. confirmed In 0-h liven, those determined theme was minimal by Western cytosolic blot staining, and only 1 to 2% of nuclei showed positive staining for the Rb protein. By 1 2 h post-PH, however, approximately 30% of nuclei were positive for Rb staining, and by 30 h, 45% of nuclei were immunoreactive for Rb antigen. In a pattern similar to that observed by immunoblot analysis, very few nuclei were moderate positive staining at 1 8, 42, and 48 h post-PH. was observed However, at 60 h, and a relatively strong signal was detected at 72 h post-PH, when approximately 25% of nuclei were immunoreactive. Cytosolic staining for Rb protein was most significant at 18 and 72 h Cell A Time 0 0 0.5 0.5 1 1 3 3 6 Post-PH 6 12 a (h) 24 18 24 30 42 -- 48 60 0 Growth & Ditterentiation ppRb 72 -4.7kb .#{149}.#{149} #{149}OOOOO28 B C 0..- a (“C 0.0 ci: #{174}L Time Post-PH Time Post-PH (h) (h) Fig. I. Rb Protein expreSsion in regenerating rat liver. Rats were subjected to 70#{176}/ PH and sacrificed at the indicated times post-PH. Nuclear protein extracts ‘vere isolated and resolved by SDS-PAGE as described in “Materials and Methods.” A: Top, immunoblot of Rh expression determined using XZ161 mAb. After transter, the I)lOt was probed with antibody and detected by the ECL method. Bottom, Northern blot analysis of Rb transcript expression. Poly(A( ‘ -enriched 10-pg samples of RNA were isolated troni livers at the indicated times post-PH, processed for blotting, and hybridized with a ‘2P-labeled 9W)-bp Bgl/ll-()xaNl fragment of the niurine Rb cDNA clone, as published previously (36). Equal lane loading was determined with a 900-bp, 2P-labeled Pstl fragment ot the rat asialoglycoprotein receptor cDNA. Right, transcript sizes. B, changes in Rb protein expression through 72 h post-PH relative to 0-h controls. Protein levels we’re densitometric ally (luantitated from the immunoblot on which equal amounts of protein were added to the lanes. C. relative changes in Rh phosphorlation (luring 72 h of re’gene’ration determined from immunoblot analysis. The results are representative of three different experiments. pRh, hypophosphorylate’d Rb; ppRb. hvpe’rpliosphorvlated Rb. post-PH. niAb F20.2F to 3-microglobulin was used as control and showed no evidence of nuclear staining (data not shown). In addition to characterizing expression of the Rb protein in regenerating liver, we were interested in comparing the results to an additional tumor suppressor gene product. Several monoclonal and polyclonal antibodies were used to detect p53 through 60 h of liver regeneration (Fig. 3). The protein was detectable in nuclei isolated from 0-h liver and mildly fluctuated until 6 h post-PH, when it increased to approximately 5-fold over baseline levels. Similar abundance was noted at 1 2 h, and by 1 8 h post-PH, expression had returned to control levels. A 3-fold increase was detected at 24 h, and a dramatic increase to greater than 40-fold was noted at 30 h. Levels returned quickly to those of baseline by 48 to 60 h post-PH. Cell Cycle-modulated tein in Synchronized Expression of Retinoblastoma HuH-7 Human Hepatoma ProCells. Based on the cyclical expression of Rb protein in the regenerating liver, we investigated in greater detail the cell cycle dependency of Rb in a synchronized proliferating hepatocyte cell. Because of the difficulties involved in synchronization of cultured primary hepatocytes, we chose to examine expression of the Rb gene product in HuH-7 cells, which have been characterized as a well-differentiated human hepatoma cell line (37). Using a modification of established methods, we were able to synchronize HuH-7 cells in G, 5, and M phases. In fact, cells arrested in G, with hydroxyurea exhibited less than 8% BrdUrd labeling, in contrast to those blocked in S phase, where 97% were labeled with BrdUrd. Rb protein abundance was examined by immunoprecipitation analysis of total cell lysate during each phase of the cell cycle, as well as 2 and 6 h after S-phase release and 4 h after M-phase release (Fig. 4A). The results indicated that Rb protein levels steadily decreased from S phase through 6 h after S-phase release, when Rb expression was approximately 15% of G1 levels (Fig. 48). Protein abundance increased in M phase and reached maximum expression 4 h after M-phase release, when it was consistently 10 to 15% greater than levels in G1. Although maximum expression of hyperphosphorylated Rh protein was detected during S-phase release, its abundance was 2 to 4 times greaten than the un/hypophosphorylated protein species throughout the cycle (Fig. 4C). It is unclear as to whether the molecular weight bands below pRb represent differentially processed Rb protein species found in the cell lystate or Rb-related proteins. It was apparent that Rb protein levels in the total cell lysate fluctuated significantly during the cell cycle of HuH-7 cells. It was important then to examine the nuclear abundance of the Rb protein species in the synchronized cells to establish whether redistribution of the protein was occurring during the various phases ofthe cell cycle. In fact, Rb abundance in nuclear extracts displayed a somewhat different pattern of expression than observed in whole cell lysates (Fig. 5A). Rb expression was almost 3-fold greater in 1465 1466 3 :-‘ ‘A. I ,,; <..#{149} ..., _‘, -.‘ . -..,. . . I fr. ‘ .4’ r p.. i [B’k S ‘p 4i , ib’ S .-.I. ! a . S (I ,. a -. S I S .5 I I ,L’ S #{149}1#{149} .b ,‘ S S S I’ll’.’. #{149}. S I. ‘ . . t ,,_4 -S I F ‘ Lv: S , S . , ._#{149}sS .4.. .- S’- #{149}, ‘. b- ‘ SI I 11.1*. 1 41:? I #{149}41P . I . ,, .1 S , #{149}s ‘ “1 #{149} .‘ -,, 1,55s S #{149},‘4.I I , .A ‘I I. t a #{149} .. ‘ : ,‘ ‘#{149}f’. * I. I P”#{231},, ‘S ,. : . . I S p. -.5 t, .-.--. ,.*.. .‘-4_ . ., u:wr; -r# S ‘:;t, S ‘t .b bF:; .\r:?:. , . >“ii”I’#{149} - h._.( I , : S’ - 1 / -4 ..-sO #{149}_#{149}I’ ‘‘ #{149}S I J. (elI Time 0.5 0 1 3 C 0 (n 12 ,. p53 B 6 Post-PH 18 A (h) 24 30 42 48 Cell 60 Gi S Cycle S2 Growth & Diffe’rentiation 1467 Phase S6 M M4 ‘, :LtIIJL B G)ce LO .0(l)C C 0 0. 08 0. . o:j1 0.25 0 0.5 1 3 Time 6 12 18 Post-PH 24 30 42 48 60 0 (h) Fig. I. ))5 1 protein (‘xpression in regenerating rat liver. Rats were subjected to 70’ PH sat ritk ed at the indicated times post-PH through 60 h. Nu lear Prot(’irl e’xtracts ss-ere’ isolated and resolve(I by SDS-PAGE as descrilwd in ‘Pslaterials and Methods.” A, immunoblot of p53 expression det’rmined using mAh Pab 240. The blot was prolwd and processed as (Ies( ribed in Fig. I . B. the relative amounts of p5 were quantitated by densitonletrk analysis and plotted on an arbitrary scale using 0-h expression as unity. TIic imn#{236}unoblot is repre’sentative ot tour experiments, using three different niAbs to p5 1. .111(1 than G and reached a nadir 6 h after S-phase (Fig. 58). Interestingly, there was a reproducible increase in M phase that dissipated after release. Phosphorylation analysis of the Rb protein in nuclei more closely resembled that observed in total cell lysate in that maximal expression of the hyperphosphorylated species occurred 2 h after S-phase release relative to G1 and returned to lower levels by 4 h after M-phase release (Fig. SC). In contrast to total cell lysate, molecular hands below pRb were not detected in protein extracts from the isolated nuclei. As with the regenerating liver, we confirmed the results from the immunoblot analyses in the synchronized HuH-7 cells with indirect immunofluorescence of Rh protein expression. Again, XZ161 provided the strongest signal and least amount of nonspecific staining. The results confirmed those obtained by Western blot analysis (Fig. 6). In G1, the majority of immunofluorescence staining was detected in the cytosol of the nonconfluent HuH-7 cells. S phase was associated with a significant increase in nuclear signaling with some cytosolic staining still detectable. The most dramatic change occurred at 6 h after S-phase release when both nuclear and cytosolic abundance of Rb staining was significantly decreased to approximately 1 5% of levels. At M phase and 4 h after M-phase release, there was reappearance of significant staining in both nuclei and cytosol. In fact, approximately 20% of the cells had significantly elevated levels of nuclear staining, although the intensity was less than that observed in S phase. A number of cells showed unique distributions of Rb protein, suggesting dynamic changes in suhcellular localization during and after release from M phase. mAh F20.2F to f3,-microglohulin was used as control and showed no evidence of nuclear staining (data not shown). Gi S S2h S6h M Cell Cycle Phase Cell Cycle Phase M4h C a) U) C S phase release Fig. 4. Total RI) protein expression in synchronized HuH-7 human liepatonia cells. Subconfluent cells were synchronized in G,, S. and M phases with hydroxyurea, aphidicolin, and noco(IaZOle, respectively, as described in “Materials and Methods.” Cells treated with aphidkolin were harvested in S phase (5) or released harvested at 2 h (S2(, in media containing and 6 h (S6(. Cells 10/ FBS and no aphidicolin incubated with nocodazole and and Colcemid were harvested in M phase (M( or released in media containing 1 0’%, FBS and harvested at 4 h (M4(. Inimunoprecipitation and Western blotting were arried out as described in “Materials and Methods .“ Cell cycle status was determined using BrdUrcl incorporation at the’ indicated time points as reomnie’nded I)y the Boehringer Mannhe’ini ( elI proliferation kit. A. immunoblot of pRI) trom synchronized HuH-7 ( ells after inimunopre ipitation of total cell lysate. B, changes in total RI) protein expression in synchronized HuH-7 cells relative to G , . Rb protein levels were densitometrically quantitated from Western blots in whkh equal amounts of protein were added to each lane. (, relative changes in Rb phosphorylation during different phases of Iii’ cell cycle determined froni immunOl)lot and clensitometric analyses. The sum of the phosphorylated state’s equals the total amount of Rb expressed at each cell cycle phase. The’ results are representative of tour different experiments. pRh, hypophosphorylated RI); ppRb. hyperphosphorylated RI). Expression of Retinoblastoma-related erating Liver and Primary Hepatocytes. Proteins in Regen- In characterizing Rb expression in regenerating rat liver with the different mAbs, we detected a previously reported 300-kDa Rbrelated protein as well as two novel, immunologically cross-reactive polypeptides (Fig. 7A). They exhibited apparent molecular masses of 1 56 and 74 kDa by SDS-gel elec- 1468 Rb Protein Expression in Regenerating A Cell Gi S Rat Liver Phase Cycle S2 S6 M M4 B C . 0 2 0.0 1 0 31 S 52h S6h M Cell Cycle Phase Cell Cycle Phase M4h C a) C/) C 21 Fig. 5. Nuclear Rb protein expression in synchronized HuH-7 cells. HuH-7 cells were synchronized in the different phases of the cell cycle as described in Fig. 4. Nuclei and nuclear protein extracts were isolated and immunoprecipitated for immunoblot analysis as described in “Materials and Methods .“ A, immunoblot of Rb from synchronized HuH-7 cells after immunoprecipitation ot nuclear extract. B. changes in nuclear RI) protein expression in synchronized HuH-7 cells relative to G,. Rh protein levels were densitometrically quantitated from irnmunoblots containing equal amounts of protein/lane. C, relative changes in Rb phosphorylation during the different analyses. protein four of the cell The’ sum cycle deterniined the phosphorylated d)f expressed at each different experiments. phorylated from immunoblot states equals cell cycle phase. The pRh, hypophosphorylated results and the total densitometric amount of Rb are representative of RI); ppRh, hyperphos- Rb. trophoresis. Nuclei were isolated from various time points through 72 h of regeneration and were processed for immunoblot analysis using different mAbs to Rb. Each of the proteins showed significant increases in nuclear abundance after the livers underwent 70% PH. p300 showed increases at 0.5 and 3 h, returned to baseline levels at 6 h post-PH, and then increased 3.5-fold over 0-h levels at 1 8 h post-PH and remained elevated through 72 h of growth (Fig. 78). p156 levels fluctuated during the first 3 h, increased approximately 6-fold at 6 h post-PH, and remained approximately 3-fold increased until 60 h post-PH, when they returned to near baseline levels. The p7’4 Rb-related protein showed the same dramatic modulation exhibited by p300 and p1 56 during the regenerative period. It decreased im- mediately after PH until 6 h, when it increased 6.5-fold above 0-h levels. Its abundance was 2- to 3-fold above baseline until 60 h post-PH, when it peaked 7-fold above baseline levels and then returned to near 0-h levels at 72 h post-PH. To further characterize the antigenic similarities between Rb and its related proteins, a series of mAbs raised against different epitopes of native Rb protein were used to map p300, p156, and p74. Monoclonal antibodies XZ161, xz1 21 , and XZ77, which were used in the immunoprecipitation and immunoblot analyses ofthe 1 1 0-kDa Rb protein, recognize epitopes 393-621 and 715-802, 444-621, and 444-535 and 620-665, respectively, in the protein (38). p300, p1 56, and p7’4 were detected by XZ1 61 in rat liver, but p74 displayed very weak antigenicity. XZ1 21 exhibited very strong affinity for p1 56, as did XZ77 for p74, but only weak affinity for the p1 1 0 Rb protein. Taken together, p1 56 expressed the putative Rb protein epitopes corresponding to aminoacids 536-621 and possibly 715-802 and 393-443 in the Rb protein; p74 appeared to share an epitope at 620-665, and p300 expressed epitopes corresponding to amino acids 715-802 and possibly 393-443 in the Rb protein. Based on the results of Rb and Rb-related protein levels in the regenerating liver, we investigated their expression in isolated hepatocytes that were maintained in culture for 96 h. We were particularly interested in comparing Rb expression to that of the novel related proteins p1 56 and p74 (Fig. 8). Under culture conditions in which the cells were incubated in 1 0% fetal bovine serum and allowed to replicate, both the pl 10 Rb protein and itsrelated proteins p1 56 and p74 exhibited very similar patterns of expression in cultured primary hepatocytes. Although the total abundance of protein did not change dramatically during 96 h, there was a reproducible redistibution of protein from the cytosol into the nucleus. Nuclear expression reached almost maximum levels by 24, 48, and 12 h for p156, p110, and p7’4, respectively. In the case of the p1 1 0 Rb protein, the major species was, in fact, in the hyperphosphorylated state. In contrast, hepatocytes cultured in serum-free media expressed primarily the hypophosphorylated species in their nuclei, which decreased to minimally detectable levels by 42 h in culture (Fig. 9). Interestingly, from 3 h on, the lower molecular weight species below pRb were again detectable, as noted previously in the synchronized HuH-7 cells (Fig. 4A). Half-Life in Cultured Determination Hepatocytes of the Retinoblastoma Protein and Regenerating Liver. It was apparent that expression of the Rb protein fluctuated significantly over relatively short periods during liver regeneration. It has been reported previously that in the regenerating rat liven, the half-life of both the canonical 4.7-kb transcript as well as the 2.8-kb transcript exhibited mRNA half-lives of approximately 40 mm, both at 0 time and 6 h post-PH (36). In addition, transcriptional activity of the Rb mRNA increased approximately 6-fold within the first 30 to 60 mm after PH and then returned to near baseline levels. The half-life of the Rb protein was obviously an important factor involved in its modulation in the regenerating rat liver. To determine the half-life of the protein in vivo, cycloheximide was used to block protein synthesis between 3 and 6 h post-PH. This time point was chosen because the abundance of Rb protein in 0-h liver was not sufficient to perform adequate Western blot and densitometric analysis. Based on the dramatic changes in Rb protein abundance C(’ll A Time 0.5 0 A. 1 3 Post-PH 6 18 (;r’th 5I tjiffer(ntiation 1469 (h) 24 30 42 48 60 72 p300 p1 56 - - - - p74 B 7 #{149}p300 0p156 Dp74 6 w #{182})14k B , -.-. ‘ *‘Pkt . : a) 0)5 . C : .C4 0 D : I I #{149}*:* ‘4fr ?::;:. ; ,- *4 - . 0 . 0.51 3 Time .- ‘ C :1 . HinLrn?dhh t ,. I 3 0 -, I 6 1824304248 Post-PH 6072 (h) Fig. 7. Expression of Rb-related proteins in regenerating rat liver. Rats were subjected to 70/ PH, and livers were harvested at the incli ated times post-PH. Nuclei and nuclear protein extracts were isolated and resolved by SDS-PAGE as described in “Materials and Methods .“ After transfer, the blots were probed with mAbs recognizing different epitopes of human RI) protein. A, immunoblots of Rh-related proteins expressed through 72 h post-PH and visualized by the ECL method. 1)300 was hybridized with XZ1 61 , p1 56 was hybridized with XZ121, and p74 was hybridized with XL77. B. (hanges in 0300, 01 56, and p74 expression through 72 h P05t4’H relative to controls. The Rb-related proteins were densitometrically quantitated from Western I)lOts containing equal quantities d)f protein/lane. The results representative of at least three different experiments. D - during the regenerative period, it was not surprising to determine that the half-life of the Rb protein in regenerating liver was 1 .9 h (data not shown). This was similar to the half-life of the protein in cultured primary hepatocytes, which was determined to be 2.2 h using an S-labeling technique and no cycloheximide (Fig. 1OA). As a comparison, the protein half-lifeofthe p1 56 Rb-related protein was 4.25 h (Fig. 108). . .‘ , ,. i_’ * - . !i’:* ,.s’. , : ?‘: - t E . .,- ,‘,. ; ...- ,‘ ‘ I ‘i ‘- Effect of TGF-1 on Retinoblastoma, E Protein Expression in Regenerating CDK4, and Cyclin Liver. It is well es- tablished that TGFinhibits the growth of certain cell types, including hepatocytes, by modulating progression through the late G, phase of the cell cycle (39). In addition, it has also been shown that i.v.-administered TGF-f31 inhibited DNA synthesis in the regenerating liver after PH (40). A candidate target for the growth-inhibitory 5... 0’: 0-h the are’ , S ‘p Fig. 6. Immunofluorescence localization of Rb antigen in sync Iironiied HuH-7 cells. Cells were synchronized into the different phases of the cell cycle as described in Fig. 4 and “Materials and Methods,” fixed in paratormaldehyde, and subjected to indirect immunofluorescence with affinity-punfied mAb XZ1 61 . RI) is distributed primarily in the cytosol (luring G , (A) and located almost entirely in the nucleus during S phase (B). C, 6 h after S-phase release, Rb is almost undetectable in the nucleus and only slightly (Iete’( table in the cytosol. In M phase )D) and 4 h (F) after M-phase release, RI) antigen is distributed in both the’ nuclear and cytosolic compartments, with parti ularly strong staining in 5OfllC nuc lei. The immunofluores e’nce distribution of Rb antigen during the difterent cell cycle phases agrees with the distnil)ution of Rb protein dlet(’rmine(I by immunol)lot analysis 0) HuH-7 cells. B,ir. pm. 25 1470 RI) Protein Expression Regenerating in Time 0 3 6 12 Rat Liver in Culture 18 24 36 significantly reduced (P< 0.001 ; Fig. 1 1). In addition, hypophosphorylated species as a percentage of total protein decreased more rapidly than the hyperphospho- (h) 48 72 84 96 ___________ 60 uclei rylated animals p156 -- - whole .-. cells -wwwww ______________ p110 #{149},a. #{149} 0 0 nuclei whole cells ______ nuclei - w p74 i#{149}i.i#{149}iii whole cells Fig. 8. Expression of RI) arid Rb-related proteins in cultured primary hepatocytes. Hepatocytes were isolated and maintained in culture with 1 0% FCS through 96 h. At the indicated times, cells were harvested and processed for total cell lysate and nuclear proteins as described in “Materials and MethO(IS.” Equal amounts of protein from each time point were loaded onto gels and processed for immunOl)lot analysis and detection of p156, pRb(110(, and p74 using mAbs XZ121, XZ161, and xZ77, respectively. Nuclear and whole lysate proteins were subjected to ECL Western blot analysis. The results are representative of three different experiments. A Time 0 0.5 1 3 6 in Culture 12 18 24 6 h in the treated expression at 6 h post-PH was not significantly different, a faint above ppRb and perhaps representing an additional perphosphonylated species consistently disappeared band hyin the b b 0 Rb protein between 1 and (P < 0.05). Although Rb the Rb TGF-j31-treated group (Fig. 1 1A). It has been reported that during the cell cycle, the Rb protein is phosphorylated under the control of certain cyclin-CDK complexes, including cyclin D-CDK4 and cyclin E-CDK2 (41 ). Based on these putative interactions, we examined the effect of TGF-31 on expression of CDK4 and cyclin F in regenerating rat liver. TGF-f31 administration dramatically inhibited the expression of CDK4 at 1 and 6 h post-PH (P < 0.001 ) and reproducibly increased levels of the protein absence of creased in between 1 at 24 h post-PH (Fig. 1 2A). Interestingly, in the the cytokine, the CDK4 protein steadily deabundance from 1 to 24 h post-PH (P < 0.01 and 6 h; P < 0.001 between 6 and 24 h). However, the levels of CDK4 associated with TGF-1 inhibition at 1 , 6, and 24 h post-PH were almost identical. Cyclin E protein expression in control regenerating liver increased approximately 3-fold between 1 and 6 h (P < (h) 30 42 48 ia#{225}f#{149} 60 72 A -pRb 100 B C 0 Cl) C 0 .o 15 cog - Ui- 0.0 . Ui0 -cD .._4 . . . . Time Time in Culture hyperphosphorylated (h) 100 C 0 Cl) Cl) each time point were loaded on gels and processed for inimunoblot analysis using XZ161 mAb. The immunoblot reactions were visualized by the ECL method. B, changes in total Rh protein expression through 72 Ii in culture relative to 0-h controls. RI) protein levels were densitometrically quantitated from imniunoblots in which equal amounts of protein were added to each lane. The results are representative of three different experiments. pRb, Rb; ppRb, Post-Chase B (h) Fig. ‘). Rb protein expression in nonreplicating cultured hepatocytes. A, hepatocytes were isolated and cultured in media without serum through 72 h as described in “Materials and Methods.” Equal amounts of protein from hypophosphorylated . 0. )( 10 LO Rb. effect of TGF-1 in replicating hepatocytes was the Rb protein. To determine whether administration of the cytokine was associated with changes in abundance and/or phosphorylation of Rb expression in the regenerating liver, TGF-j31 was iv. administered 45 mm prior to sungery and 1 2 h after PH at a dose sufficient to inhibit DNA synthesis. Although it has been shown previously that TGF-pl administration had little effect on transcript expression, Rb protein expression at 24 h post-PH was o (0 Time 1 0 - 0. Post-Chase 2 4 6 - 2 Fig. 10. Rb protein and p156 4 (h) 8 10 - 6 Time Post-Chase half-life determinations 8 10 (h) in cultured primary hepatocytes. Isolated primary hepatocytes were pulse-chase labeled with ltrans-35Slmethionine. At the indicated times, the cells were harvested, lysed, and incubated with XZ161 for pRb and XZ121 for p156. The immunoprecipitated Rb protein (A) and p1 56 (B) were analyzed by SDS-PAGE and subjected to densitometnic analysis as described in “Materials and Methods.” The apparent half-lives were determined by linear regression analysis. Cell A Time Post-PH 1 24 :.. I B staining tocyte C 0Cl)’- .0 1- +11- l 1 24 6 Time Post-PH & Differentiation 1471 and corresponds to the peak of delayed-early gene expression (34). After a dramatic decrease just prior to peak S phase, Rb reaches its highest levels at 24 to 30 h post-PH, corresponding to peak DNA synthesis and initiation of the first wave of mitosis. A similar but less abundant cycle of Rb expression also occurs during the second mound of cell proliferation in the regenerating liven. Very similar findings were determined by immunocytochemistry in which Rb protein expression was identified by immunofluorescence (h) 6 Growth (h) Fig. 1 1. Effects oITGF-f31 on Rb protein expression in regenerating rat liver. Animals were injected iv. with vehicle or 1 0 pg ofTGF-f31 45 mm before PH and 1 2 h post-PH. A, immunoblot analysis of Rb protein expression in nuclei isolated from 1 -, 6-, and 24-h regenerating liver treated with vehicle (-) or TGF-f31 (+) as described in “Materials and Methods.” B, densitometric quantitation of changes in total Rb protein expression and phosphorylation status from 1 -, 6-, and 24-h post-PH regenerating livers relative to 0-h controls. Equal amounts of protein were added to each lane of the immunoblots. The results are representative of three different experiments. pRb, hypophosphorylated Rb; ppRb, hyperphosphorylated Rb. of the regenerating nuclei that stained liven. The percentage of hepa- positively for Rb at 24 to 30 h post-PH is almost identical to the percentage of cells undengoing DNA synthesis and mitosis (32) and supports the observation by Western blot analysis that Rb protein expression and cellular localization are cell cycle regulated. The results are consistent with the notion that the Rb gene plays a key role in the regulation of the cell cycle (28). However, the data also suggest a significant uncoupling of transcript and protein expression for the tumor suppressor gene. It has been shown previously that major changes in Rb transcript expression occur only through the first 1 2 h in the regenerating liven (36). Although the total abundance of mRNA does not change during that period, theme is a significant shift in expression between the 4.7- and 2.8-kb species. No additional changes in transcript expression or transcriptional mate occur at times when peak protein expression is observed. Interestingly, the p53 tumor suppresson gene exhibits similar patterns of both transcript and protein expression following PH as does Rb. Peak transcript expression occurs during G1 at 6 h post-PH (data not shown), and similar to Rb, the major peak in protein ex- pression and then decreased 60% (P< 0.001) at 24 h post-PH (Fig. 1 28). TGF-1 administration was associated with significant decreases, as great as 90%, in protein expression at each time point (P< 0.001). 0.01) Discussion The liven is unique in its ability to regenerate. It represents a remarkable in vivo model for the study of gene expression and growth regulation. Within minutes after PH, numerous cellular changes take place that prime hepatocytes to replicate. The process involves a complex pattern of gene expression and a modulation of numerous transcripts duning the growth period (33, 34). Although the immediate- and delayed-early genes are probably responsible for hepatocytes to transition from G0 to G1, many other cell cycle- and growth-regulated genes are responsible for the progression examining through expression mitosis. This is the first detailed of the Rb tumor suppressor product in regenerating hepatocytes. In addition, hepatoma cell line, Rb protein cell cycle dependent Rb expression Hepatocytes liven as well as primary using a well-differentiated abundance and exhibited similar in an essentially maintain capacity a remarkable presented in this study indicate liver expresses low levels of Rb increase in Rb protein expression erating state after 70% PH. The 1 2 h post-PH occurs within the gene cultured human was shown to be characteristics in the in vivo liven regeneration of the adult liven are long-lived ated cells that remain report quiescent to model. diffenenti- state but to proliferate (33). The data that the normal adult mat protein and that a marked occurs during the megenfirst significant increase at G1 phase of the cell cycle is at 30 h post-PH. This parallel pattern of expression for two very different tumor suppressor genes appears to be more than coincidental and may reflect their involvement in the first rnitotic wave after PH as a checkpoint for further proliferation. In this regard, a recent report demonstrates a direct link between the two proteins in controlling cell growth and apoptosis (42). In support of the apparent uncoupling between protein and mRNA levels, cytosolic levels of p53 and Rb protein in the regenerating liver, although less abundant, show a similan pattern of modulation to that exhibited in nuclei (data not shown). Theme is a growing list of genes, in fact, in which steady-state transcript expression is uncoupled from protein expression (43). In this regard, it has been shown that selective translational control of mibosomal protein mRNAs constitute an important regulatory mechanism opemating in vivo in the course of liven regeneration (44). Theme has been some controversy as to whether abundance of the Rb protein is modulated during the cell cycle. It has been reported that no significant differences were detectable in the staining pattern on distribution of the Rb protein in the G1, S, and G2 phases of the cell cycle in a collection of Rb-expressing cell lines (45). In contrast, the apparent lack of nuclear staining in a group of Rb-positive tumor cells resulted from a significant decrease in total cellular Rb protein during G0 on middle G1 (46). Progression of embryonic stern cells towards the G1-S transition was similarly accompanied by a marked decrease in total abun- dance of Rb protein (47). In addition, differentiation of the embryonic stem cells was associated with a marked increase in total amounts of Rb protein as observed previously when embryonal carcinoma cells were induced to differentiate into neumoectodemmal cells (48). The results of the present study indicate that in regenerating mat liver and 1472 Rb Protein Expression in Regenerating Rat Liver B A Time Post-PH 1 - Time (h) 6 + - I 24 + - Post-PH (h) 6 Fig. 12. Effects of TGF-(31 on CDK4 and cyclin E protein expression in regenerating liver. Immunoblot analysis and densitometric quantitation ofCDK4 (A) and cyclin E (B) expression in regenerating liver treated with TGF-j31 were performed as described in Fig. 1 1 and “Materials and Methods.” Densitometric quantitation of changes in 24 + Cyclin E CDK4 C C 0 (I) 0 Co. nuclear protein expression from 1 -, 6-, and 24-h post-PH regenerating livers was expressed relative to 0-h controls. Equal quantities of protein were loaded onto each of the lanes. Representative immunoblots from three different experiments are shown. 0. 0 0 > 0 Time Post-PH Time Post-PH (h) HuH-7 human hepatoma cells, the total amount of Rb protein changes dramatically during the various phases of the cell cycle. This may be facilitated, in part, by the nelatively short half-life ofthe Rb protein in both systems. In the hepatoma cell line, maximal decrease in total cellular Rb protein occurred subsequent to S-phase release and prior to M phase. In contrast, the quiescent liven representing a unique in vivo example of the G0 state of the cell cycle exhibited almost undetectable levels of Rb protein. It is well documented that the state of phosphonylation of the Rb protein fluctuates during the various phases of the cell cycle (7, 49). Generally, in G0 and early G1, Rb protein exists primarily in the hypophosphorylated form. As the cells transit into mid/late G), the protein undergoes additional phosphorylation and then remains in this hypemphosphorylated form throughout S phase, G2, and most of M phase (50). In short, underphosphorylated Rb protein appears to block passage through the G1-S boundary of the cell cycle; hypemphosphorylation relieves the block and allows cell replication to occur. It was, therefore, somewhat surprising that the Rb protein remained primarily in the hypemphosphonylated state in regenerating liven and cultuned primary hepatocytes. In contrast, primary hepatocytes cultured in serum-free media expressed primarily the hypophosphonylated form of the Rb protein. It was also interesting that a significant portion of the Rb protein during the G1 block in HuH-7 cells was hypenphosphonylated. Howeven, it has been reported previously that cells growtharrested in G1 with hydroxyunea exhibited hypemphosphomylation of Rb protein (51 ). Our results support the recent observation that substantial phosphorylation of Rb exists in G1 even prior to the hypemphosphorylation point, suggesting the existence of distinct patterns of phosphorylation that are associated with different subsets of Rb protein molecules (52). In these hepatocyte models of cell growth, the phosphonylation of Rb is not coordinated with the G1-S transition and may not directly regulate it. The fact that most of the Rb protein was phosphorylated in regenerating liver and highly proliferating HuH-7 cells implies that in these replicating models, the hyperphosphonylated Rb has lost its ability to interact with a variety of nuclear proteins. How- (h) even, although the loss of binding to proteins such as transcmiption factor E2F is assumed to reflect a functional mactivation of Rb it may, in fact, permit additional functions of the Rb protein. For example, a recent report demonstrated that the COOH-temminal domain of Rb, outside of the NB pocket, complexes and regulates the activity ofc-abl, which has been shown to phosphorylate the catalytic subunit of RNA polymemase II (53). Furthermore, the c-AbI-Rb cornplex is disrupted by phosphorylation of the Rb protein during the cell cycle. Theme is a growing family of proteins that shame structural similarity with the Rb protein. Two of those proteins, p107 (54) and p1 30 (55), were isolated by their interaction with the region of adenovinus E1A that binds Rb. In addition, p300 also shows immunological cross-reactivity to the vanious subsets of Rb protein (38), although its binding site to adenovirus El A is distinct from that of Rb, p1 07, and p130 (56). In the present study, two additional and novel immunologically cross-reactive proteins, p1 56 and p74, were identified in regenerating liven by a series of mAbs against human Rb protein (38). In subsequent experiments, both proteins could be immunoprecipitated from three human hepatoma cell lines (HepG2, HuH-7, and Hep3B), Ads transformed primary human embryonal kidney cells 293, and human osteogenic sarcoma cells (Saos-2) but not from African green monkey kidney cells (CV-1 ), or an immortal- ized mouse hepatocyte cell line (AML-l Both proteins exhibited significant pression during liven regeneration primary hepatocytes. p74 exhibited bution as p1 56 except 2; data not shown). induction as well a similar that it was additionally in nuclear exas in cultured cellular distni- detected in cells (data not presented). However, tryptic digests of the isolated proteins indicated that they are different and that p1 56 is not simply a dimem of p74 (data not shown). The precise moles of p156 and p74 in cell growth remain to be determined. However, it is interesting that p1 56 and p74 are present in the Rb protein-deficient Saos-2 and Hep3B cells. The results suggest that both Rb-related proteins may substitute certain functions of Rb protein in the control of cell proliferation and differentiation. COS-7 Cell The mechanism by which TGF-l inhibits cell prolifenation is poorly understood and probably involves the interplay of a number of gene products. For example, it has been reported that TGF-l suppresses c-myc gene transcription by modulating the binding of cellular factors, including Rb, to the 5’ regulatory region of the gene (57, 58). More recently, the mechanism of TGF-l inhibition has been related to its ability to prevent hyperphosphorylation of Rb protein through its effects on expression of Gl cyclmns and their associated cyclmn-dependent kinases (59). It has been shown previously that TGF-31 induces transient inhibition of liver regeneration in rats and mice (40, 60) in the absence of changes in Rb transcript expression (36). However, the present study indicates that TGF-l not only inhibits Rb protein phosphonylation in cultured primary hepatocytes (data not shown) but also inhibits Rb protein expression in the regenerating liver. The results also indicate that TGF-l induces significant decreases in both CDK4 and cyclin E as early as 1 h post-PH. In this regard, it has been reported recently in Mvl Lu mink lung epithelial cells that TGF-3l induced suppression of CDK4 synthesis during G1 (61 ) and inhibited cyclin E-associated kinase activity (62). Moreover, in Mvl Lu mink lung epithelial cells, TGF-f31 functions in another manner by raising the threshold level of cyclin E necessary to activate CDK2 through an inhibitor that binds cyclin E-CDK2 complexes (63). Inhibition of CDK4 synthesis by TGF-f31 is linked to G1 arrest and probably involves a collaboration of Gi cyclins in the functional inactivation of the Rb protein (64, 65). These data suggest that TGF-f31 inhibits Rb protein phosphorylation, at least seemingly by suppressing expression of CDK4 and cyclin E, resulting in a decrease of cyclin D-CDK4 and cyclin E-CDK2 complexes. Furthermore, it was reported recently that the cell cycledependent expression of cyclin Dl is dependent on the presence of functional Rb protein (66). Our results suggest that the effect of TGF-l on the phosphorylation status of Rb in the regenerating liven may involve numerous factors as well as the total abundance of the protein. In conclusion, the results of the present study indicate that the regenerating mat liven represents a remarkably unique in vivo system for studying cell cycle expression of the Rb tumor suppressor gene product. It provides an opportunity for examining the function of the Rb protein in normal cell growth and differentiation of cells in the whole organism. It is attractive to consider, for example, that p1S6 and p74 are similar enough to Rb that the three proteins shame similar functions in vivo. Future studies will undoubtedly provide us with the necessary information to establish their own role as potential tumor suppressors. Factors controlling the regeneration of an entire organ are obviously complex. For example, the pattern of Rb protein levels during the first round of cell replication indicates a significant uncoupling of transcript expression and translation. Interestingly, a similar uncoupling of mRNA and protein expression was also observed for the p53 tumor suppressor gene (data not shown). Future studies will provide impomtant information regarding the role of translational-dependent expression of these tumor suppressor genes and their role in modulating hepatocyte growth and differentiation. Materials Materials. and Methods mAbs XZ1 61 , XZ1 21 , and XZ77 to human Rb protein and Pab 242 and 421 to human p53 were genenously provided by Dr. Ed Hanlow (MGH Cancer Center, Growth & Differentiation Chanlestown, MA). mAb F20.2F to j32-microglobulin was kindly provided by Dr. Ronald P. Messnen (University of Minnesota Medical School, Minneapolis, MN). Goat antimouse and anti-rabbit IgG horseradish penoxidase were purchased from Bio-Rad Laboratories (Hercules, CA). Nonmal goat serum and goat anti-mouse IgG Cy3 conjugates were purchased from Jackson ImmunoReseanch Labonatonies, Inc. (West Grove, PA). Protein A-Sephanose 6 MB was obtained from Pharmacia Biotech, Inc. (Piscataway, NJ). mAbs against cyclin E (HE-i 2) and p53 (Pab 240) and rabbit polyclonal antibody to CDK4 were purchased from Santa Cmuz Biotechnology, Inc. (Santa Cnuz, CA). Cell cycle synchronization reagents and Hoechst dye were purchased from Sigma Chemical Company (St. Louis, MO). All other standard reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI), Curtin Matheson Scientific (Eden Praine, MN), on Fisher Scientific (Itasca, IL). Animals and Surgical Procedures. In brief, male Sprague-Dawley mats (250 to 275 g) were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN), maintained on a standard 12-h light/dark cycle, and fed commercial laboratory chow ad libitum. They were subjected to midventral lapanotomy and 70% PH under ether anesthesia between 9 a.m. and 1 1 a.m., as described previously (31). At various times after PH, the animals were sacrificed, and the remnant livers were removed and rinsed in normal saline solution; then a 0.5-cm cube from the night lower lobe was excised and embedded in OCT (Baxter Scientific, Minneapolis, MN) for immunohistology. The remaining liver was flash-frozen in liquid nitrogen. Sham control livers were obtained under similar conditions but without PH. To inhibit protein synthesis, a dose of 5 mg/i 00 g body weight of cycloheximide (Sigma) was administered i.p. 1 h before surgery (36). Ten pg of TGF-(31 (R & D Systems, Inc., Minneapolis, MN) were administered as an i.v. bolus 45 mm prior to surgery and 1 2 h after PH. All animals received humane came in compliance with the institute’s guidelines as outlined in “Guide for the Cane and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the NIH (NIH publication number 86-23, revised 1985). Isolation of Hepatocytes and Cell Culture. Rat hepato- cytes were isolated by standard collagenase perfusion as described previously (67). The hepatocyte suspensions were obtained by filtering the digested livers through 5evemal layers of gauze to remove undigested material. The cells were then centrifuged at 50 x g for 2 mm, and the supennatant was removed by gentle aspiration. After filtration through a single layer of gauze, the cells were washed in modified Eagle’s medium; cell viability was 85 to 90%, as determined by trypan blue exclusion. The freshly isolated hepatocytes were plated on 1 00-mm Pnimania tissue culture dishes (Becton Dickinson Labware, Lincoln Park, NJ) at 7.5 x i0 cells/cm2 in Williams’ E medium (GIBCO, Grand Island, NY) supplemented with 26 mi sodium bicarbonate, 23 mi HEPES, 0.01 uniVml insulin, 2 mi i-glutammne, 10 nM dexamethasone, 5.5 mrvi glucose, 1 00 units/mI penicillin, and 100 units/mI streptomycin. Cultures were maintamed at 37#{176}C in a humidified atmosphere of 5% CO2. After 2 h, the cultures were washed with fresh media to remove dead cells and loosely attached aggregates. The medium was changed every 24 h by gentle pipetting. To stimulate growth, 1 0% heat-inactivated FCS (Atlanta Biologicals, Inc., Noncross, GA) was added to the medium. Hepatocytes were harvested at times indicated in the figure legends, flash- 1473 1474 Rb Protein Expression in Regenerating Rat Liver frozen in liquid nitrogen, hepatocellular carcinoma in DMEM supplemented icillin, and 100 units/mI and stoned at -70#{176}C.The human cell line HuH-7 was maintained with 1 0% FCS, 1 00 units/mI penstreptomycin. Cell Cycle Synchronization. HuH-7 cells were synchronized as described previously (47) with modifications. In brief, subconfluent cells were starved for 24 h in media containing 0.i% FBS. The cells were then washed and incubated for 1 8 h in media containing 1 0% FBS and either 2.5 pg/mI aphidicolin to establish S phase on 0.1 mri hydnoxyunea to synchronize in G1 . Cells treated with aphidicohn were washed twice and incubated in fresh media with 1 0% FBS for 2 on 6 h. For M-phase synchronization, cells were incubated for 1 6 h in media containing 1 0% FBS and 80 np/mI nocodazole; 0.06 igJml Colcemid was added to the media for an additional 2-h incubation. Cells were washed twice in media with 1 0% FBS and harvested immediately (M) or after 4 h of incubation (M4). The cells were pelleted, flash-frozen in liquid nitrogen, and stored for Western blot analysis at -70#{176}Cfor no longer than 3 days. Cell cycle phases were determined by BndUnd inconponation at various times using a cell proliferation kit and the manufacturer’s recommendations (Boehninger Mannheim Corp., Indianapolis, IN). The labeled cells were washed in PBS and fixed in 75% ethanol at -20#{176}Cfor 30 mm. The dishes were washed twice in PBS containing 0.1% Tween 20 and incubated in PBS containing 5% normal goat serum to reduce nonspecific binding. The BndUnd-Iabeled cells were stained with anti-BmdUnd mAb supplemented with DNase and sequentially incubated with anti-mouse-immunoglobulmn-fluonescemn. Total DNA was stained with Hoechst dye 33258 (Sigma). Immunolabeling was analyzed using a Zeiss standard fluorescence microscope (Carl Zeiss, Inc., Thonnwood, NY). Photographs were taken with Kodak Ektan-l 000 film (Eastman Kodak Co., Rochester, NY). At least 300 cells/time point were counted for evaluation of BmdUnd incorporation. Gel Electrophoresis and Immunoblotting. Rb protein was cells immunoprecipitated from total lysate by incubating in 1 ml lysis buffer containing 250 msi NaCI, 0.1% NP4O, 50 mM HEPES (pH 7.0), 5 mM EDTA, 50 mr’i NaF, 0.1 mM sodium orthovanadate, 50 pg phenylmethylsulfonyl fluonide, 1 jg leupeptin, 1 pg aprotinin, and 1 mi DII for 30 mm on ice. Cell debris was removed by centnifugation at 1 0,000 x g for 1 mm. The lysate was precleaned with 40 p1 of normal goat serum and 1 00 p1 of fixed, killed Staph ybcoccus aureus cells (Zymed Laboratories, Inc., South San Francisco, CA) and then incubated with 1 00 p1 of cultured hybmidoma supennatant overnight at 4#{176}C. The lysate was then incubated with Protein A-Sepharose for 2 h at room temperature. The Sephanose beads were washed three times with lysis buffer and then incubated in denaturing buffer at 95#{176}C for 3 mm for immunoblot analysis. Nuclei were isolated from liver tissue and cultured cells as described previously (68, 69). Nuclear proteins were isolated and immediately flash-frozen as small aliquots in liquid nitrogen or electrophonesed immediately on 6% SDS-polyacrylamide gels. After electrophonetic transfer to nitmocellulose membranes, the blots were incubated with i5% hydrogen peroxide for 1 5 mm. The blots were probed with specific primary mAb after residual protein binding sites were blocked with 5% milk in Tnis-buffemed saline (TBS). After several washes in TBS, the blots were incubated with goat anti-mouse antibody for 1 h, and protein was visualized by ECL detection (Amensham Corp., Arlington Heights, cord i ng to the manufacturer’s recommendations. RNA Isolation and Northern Blot Analysis. IL) ac- Total RNA was isolated from liver tissue as described previously (70). PoIy(A)-enmiched RNA, obtained by oligo-dT (New England Biolabs, Beverly, MA) chromatography was electrophomesed on 1 % agamose, 2.2 M formaldehyde, 1 x 4-mompholinpmopanesulfonic acid (pH 7.0) denaturing gels and transferred by passive capillary diffusion to MagnaGmaph nylon membrane (Micron Separations, Inc., Westbomo, MA). The large molecular weight RNA ladder from BRL (Bethesda Research Labs, Gaithersbung, MD) was used for nucleic acid standards. A 960-bp BgblI-OxaNI fragment of the mouse Rb gene (71) and a 900-bp Pstl fragment of the mat asialoglycoprotemn receptor gene (72) were labeled with [a-32P]dCTP (3000 Ci/mmol; Amersham Corp.) by random priming (73) using a commercial kit (United States Biochemical Corp., Cleveland, OH). Hybridizations were penformed for 1 8 to 24 h at 42#{176}C, as described previously (36). Membranes were washed twice for 1 5 mm at room ternpematune in 1 X SSPE-0.l% SDS and twice at 42#{176}Cin 1 X SSPE-0.5% SDS. Autoradiography was done with Kodak XAR film (Eastman Kodak Co.) at -70#{176}Cusing an intensifying screen. Densitometric Analysis. Video densitometny was penformed using a Macintosh II (Apple Computer, Cupertino, CA) coupled to a Data Translation DT2255 video digitizer (Data Translation, Marlboro, MA) and a JVC GX-N8 video camera (JVC Corporation of America, Elmwood Park, NJ), as described previously (74). Quantitation of autonadiograms and fluonograms used the NIH Image 1 .4 Densitometric Analysis Program. Statistical analysis was performed using InStat version 2.01 to calculate ANOVA and Bonfenmoni multiple compamisons test P values. Immunocytochemistry. from control and OCT (Baxter Scientific). Sample regenerating Cryostat tissues mat livers sections were and collected embedded 5 pm-thick in were cut, mounted on slides, fixed for 5 mm in 4% buffered (pH 6.9) pamafonmaldehyde containing 15% (v/v) saturated picnic acid, and incubated with 5% normal goat serum in TBS for 2 h. Sections were then incubated with the Rb mAb XZ161 or mAb F20.2F against 32-rnicroglobuImn in 0.3% Triton X-l 00/PBS overnight. After three washes, the slides were incubated with a secondary antibody (cyanine 3.18conjugated goat anti-mouse IgG; Jackson ImmunoReseanch) in 0.3% Triton X-iOO/PBS (1 :200) for 1 h and then countenstained for cell nuclei in a solution of 1 pg/mI of Hoechst dye 33258 for S mm. Sections were examined using an Olympus BH2 microscope equipped for vertical dankfield illumination using a mercury lamp (Olympus, Lake Success, NY). Hoechst 33258 was visualized using a Schott UG1 exciter filter and a 420-nm Iongpass emission filter; cyanine 3.18 was visualized using a 541-551-nm exciter filter and a 573-607-nm emission filter. Colon photographs were made using Kodak Ektam-l000 films (Eastman Kodak Co.). Synchronized HuH-7 cells were fixed and processed in a similar fashion. Protein Half-Life Determinations. The in vitro Rb protein half-life was determined in hepatocytes that were isolated from control livers and grown on 3.5-cm Pmimania culture dishes (Falcon #3801) as described above. After 24 h in culture, the cells were washed in serum-free media and then incubated in 2 ml of methionine-free DMEM containing 0.2 mCi/mI of trans-355 label (ICN Biomedicals, Inc., Cell Costa Mesa, CA) for 4 h at 37#{176}C. The radioactive medium was removed, and the cells were washed several times with Williams’ E medium containing 10% FBS and then with chase medium containing 1 5 mg/mI of methionine and 10 mg/mI of cysteine and harvested at the indicated time points. Proteins were normalized to total cell protein content and analyzed by immunoprecipitation and electmophonesis. Gels were dried and subjected to automadiogmaphy and quantitated by densitometry as described previously (36). The in vivo half-life of the Rb protein was assessed in the regenerating liven 3 to 6 h post-PH by inhibiting protein synthesis with cycloheximide at the 3-h time point post-PH. The decay rate of the Rb protein was determined on immunoblots using video densitometry as described above. Because Rb was almost undetectable in 0-h liven, the 3-h time point after PH was designated as the initial decay point from which to determine the half-life. Remnant Rb protein at 3+n h after decay was calculated by standard regression analysis using the formula [fts3+nhPH ft”3+nhPH] [fts6hPH _ ft6hPi-i], where t represents time, s represents protein synthesis, d represents protein degradation, and PH is post-partial hepatectomy. Acknowledgments We are especially grateful to Dr. Ed Harlow and Chidi Ngwu (Massachusetts General Hospital, Harvard Medical School, Charlestown, MA) for mAbs to the Rb and Rb-related proteins. We also thank Drs. Cary Mariash and Yuichiro Sudo for isolation of the primary hepatocytes, Dr. Richard Stockert (Albert Einstein College of Medicine, Bronx, NY) for the hepatoma cell line HuH-7, Dr. Wen-Hwa Lee (University of Texas Health Science Center, San Antonio, TX) for helpful discussions, and Janeen Trembley for critical reading of the manuscript. References 1 . Friend, S. H., Bernards, R., Rogelj, S., Weinberg, R. A., Rapaport, Albert, D. M., and Dryja, T. P. 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